Papillomaviruses (PV) have been linked to widespread, serious human diseases, especially carcinomas of the genital and oral mucosa. Tens of millions of women suffer from human papilloma virus (HPV) infection of the genital tract. Significant number of these women eventually develop cancer of the cervix. It has been estimated that perhaps twenty percent (20%) of all cancer deaths in women worldwide are from cancers which are associated with HPV. As many as 90% of all cervical cancer maybe linked to HPV.
Papillomaviruses also induce benign, dysplastic and malignant hyperproliferations of skin and mucosal epithelium (see, for example, Mansur and Androphy, (1993) Biochim Biophys Acta 1155:323-345; Pfister (1984) Rev. Physiol. Biochem. Pharmacol. 99:111-181; and Broker et al. (1986) Cancer Cells 4:17-36, for reviews of the molecular, cellular, and clinical aspects of the papillomaviruses).
HPV""s are a heterogeneous group of DNA tumor viruses associated with hyperplastic (warts, condylomata), pre-malignant and malignant lesions (carcinomas) of squamous epithelium. Almost 70 HPV types have been identified, and different papillomavirus types are known to cause distinct diseases, c.f., zur Hausen, (1991) Virology 184:9-13; Pfister, (1987) Adv. Cancer Res., 48:113-147; and Syrjanen, (1984) Obstet. Gynecol. Survey 39:252-265. HPVs can be further classified either high risk (such as HPV type 16 [HPV-16] and HPV-18) or low risk (e.g., HPV-6 and HPV-11) on the basis of the clinical lesions with which they are associated and the relative propensity for these lesions to progress to cancer. For example, HPV types 1 and 2 cause common warts, and types 6 and 11 cause warts of the external genitalia, anus and cervix. HPV""s can be isolated from the majority of cervical cancers, e.g., approximately 85 to 90% of human cervical cancers harbor the DNA of a high-risk HPV. Types 16, 18, 31 and 33 are particularly common; HPV-16 is present in about 50 percent of all cervical cancers.
The biological life cycle of the papillomaviruses appears to differ from most other viral pathogens. These viruses are believed to infect the basal or germ cells of the epithelium. Rather than proceeding to a lytic infection in which viral replication kills the cell, viral DNA transcription and replication are maintained at very low levels until higher strata of the epithelium are achieved. There, presumably in response to differentiation-specific signals, viral transcription accelerates, DNA synthesis begins and virion assemble occurs.
In HPV-positive genital cancers, the viral genomes are transcriptionally active, and two viral genes, E6 and E7, are invariably expressed. The high-risk HPVs encode two oncoproteins, E6 and E7, whose expression can extend the life span of squamous epithelial cells, which are a normal host cell for the papillomavirus. E6 and E7 together can result in the efficient immortalization of primary human cells (Hawley-Nelson et al., (1989) EMBO J., 8:3905-3910; Mxc3xcnger et al., (1989) J. Virol., 63:4417-4421; Watanabe et al., (1989) J. Virol., 63:965-969). E6 and E7 are expressed in HPV-positive cervical cancer-derived cell lines (Schneider-Gxc3xa4dicke et al., (1986) EMBO J., 5:2285-2292; Schwarz et al., (1985) Nature, (London) 314:111-114; Smotkin et al., (1986) Proc. Natl. Acad. Sci. USA, 83:4680-4684). Furthermore, although many genetic changes have occurred in cervical carcinoma cells, the continued expression of the viral oncoproteins is necessary since expression of antisense E6/E7 RNA results in decreased cell growth (von Knebel-Doeberitz et al., (1988) Cancer Res., 48:3780-3785). Similar to the transforming proteins of the other small DNA tumor viruses, simian virus (SV40) and adenovirus, the transforming properties of the E6 and E7 oncoproteins appear to be due at least in part to their capacity to functionally inactivate the p53 and the retinoblastoma (pRB) tumor suppressor proteins. The E6 proteins of HPV-16 and HPV-18 can complex and cause ubiquination-dependent degradation of p53 (Werness et al., (1990) Science, 248:76-79; Schiffaer et al., Cell 75:495-505 (1993)). The high-risk HPV E7 proteins bind pRB more efficiently than the E7 proteins of low-risk HPVs (Barbosa et al., (1990) EMBO J., 9:153-160; Dyson et al., (1989) Science, 243:934-937; Mxc3xcnger et al., (1989) EMBO J., 8:4099-4015). It is believed that the functional inactivation of both p53 and pRB, and related regulatory pathways, by E6 and E7 are important steps in cervical carcinogenesis.
One characteristic of HPV-related carcinogenic progression is the frequent integration of the viral genome into the human chromosome in the cancer cells in a manner that results in the loss of expression of the viral E2 gene but maintains high levels of E6/E7 expression (Durst et al., (1985) J. Gen. Virol., 66:1515-1522; Jeon et al., (1995) Proc. Natl. Acad. Sci. USA, 92:1654-1658). The product of the E2 open reading frame plays an important role in the complex transcriptional pattern of the HPV""s. The E2 transcriptional activation protein (xe2x80x9cthe E2 proteinxe2x80x9d) is a trans-acting factor that activates transcription through specific binding to cis-acting E2 enhancer sequences in viral DNA (Androphy et al., (1987) Nature, 324:70-73), and has been shown to induce promoter expression in a classical enhancer mechanism (Spalholz et al., (1985) Cell 42:183-91). The E2 gene product exerts trans-regulatory effects in the upstream regulatory region (xe2x80x9cLCRxe2x80x9d) of the viral genome, disruption of E2 is thought to alter regulation of expression of E6 and E7 genes.
As with other transcription factors, the functions of the E2 proteins appear to be localized in discrete domains (Giri et al., (1988) EMBO J., 7:2923-29). The E2 amino terminus encompasses the transcriptional activation domain and binding site for the papillomavirus E1 replication protein. The E2 C-terminal domain is well conserved among the papillomaviruses, and contains the dimerization and DNA binding activities of E2. This domain sponsors sequence-specific interaction with DNA containing the sequence ACC(G)NNNN((C)GGT and represses the papillomavirus early promoter that drives expression of E6 and E7 (e.g., the P97 promoter of HPV 16 and the P105 promoter of HPV18). This is due to the position of E2 binding sites within the promoter: two of the four E2 binding sites within the P97 and P105 promoters immediately flank the TATA box and promoter proximal SP1 sites of these promoters, rendering them inaccessible to needed transcription factors.
The upstream regulatory region (or long control region (LCR)) is found immediately 5xe2x80x2 to the early genes of bovine papilloma viruses (BPV""s) and other papillomaviruses. The LCR contains cis-acting regulatory signals, including an origin of DNA replication and several promoters that function in early transcription. The LCR also contains enhancer elements that activate transcription from the URR promoters and heterologous promoters (Sousa et al., (1990) Biochemica et Biophysica Acta 1032: 19-37).
The E2 enhancer elements are conditional, in that they stimulate transcription only when activated by a protein encoded by the E2 open reading frame (Romanczuk et al., (1990) J. of Virol. 64:2849-2859). Gene products from the E2 gene include the full-length transcriptional activator E2 protein and at least two truncated versions of the E2 protein BPV1 that function as transcriptional repressors. Transcriptional activation and repression of viral genes by E2 gene products constitute critical regulatory circuits in papillomavirus gene expression and DNA replication (reviewed in McBride et al., (1991) J. Biol. Chem. 266:18411-18414). Within the LCR, transcriptional regulation by the E2 protein depends on its direct binding to the nucleotide sequence 5xe2x80x2ACC(G)NNNN(C)GGT3xe2x80x2 (SEQ ID NO:9) (Androphy et al., supra; Dartmann et al., (1986) Virology, 151:124-30; Hirochika et al., (1987) J. Virol, 61:2599-606; P. Hawley-Nelson et al., (1988) EMBO J., 7:525-31; McBride et al., (1988) EMBO J., 7:533-39; McBride et al., J. of Biol. Chemistry 266:18411-1844 (1991); Demeret et al. J. Virol. 71:9343-9349 (1997); Desaintes et al. EMBO 16:504-514 (1997); Thierry et al. New Biol. 10:4431-4437 (1990); and Bernard et al. J. Virol. 63:4317-4324 (1989)).
European patent application 302,758 refers to the use of modified forms of E2 protein that bind to, and block, E2 binding sites on papillomavirus DNA without resulting in trans-activation. That application also refers to repression of E2 transcriptional activation through the use of DNA fragments that mimic E2 binding sites, and thus bind with E2 trans-activators, making them unavailable for binding to E2 sites on the viral DNA.
U.S. Pat. No. 5,219,990 describes the use of E2 trans-activation repressors which interfere with normal functioning of the native full-length E2 transcriptional activation protein of the papillomavirus. However, the E2 trans-activation repressors of the ""990 patent are proteins that dimerize with the full-length native E2 protein to form inactive heterodimers, thus interfering with the formation of active homodimers comprising full-length native E2 polypeptides and thereby repressing papillomavirus transcription and replication. The E2 trans-activation repressors are described as fragments of the E2 polypeptide in which the dimerization function has been separated from its DNA binding function, e.g., the E2 trans-activation repressors includes at least the dimerization region, but less than the DNA binding domain, of the E2 polypeptide.
One aspect of the present invention provides a method of treating, e.g., lessening the severity or preventing the reoccurrence of, a papillomavirus-induced condition. In general, the subject method comprises administering to an animal, e.g. a human, infected with a papillomavirus a pharmaceutical preparation comprising a therapeutically effective amount of either (i) an E2ad/db polypeptide or (ii) a gene construct for expressing the E2ad/db polypeptide. As described in further detail below, the E2ad/db polypeptide includes a DNA binding domain and a transcriptional activation domain derived from one or more E2 proteins. The E2 polypeptide or gene construct is formulated in the pharmaceutical preparation for delivery into PV-infected cells of the animal.
In preferred embodiments, the subject method is used to treat a human who is infected with a human papillomavirus (HPV), particularly a high risk HPV such as HPV-16, HPV-18, HPV-31 and HPV-33. However, treatment of low risk HPV conditions is also specifically contemplated.
In certain preferred embodiments, the DNA binding and transcriptional activation domains of the E2ad/db polypeptide have amino acid sequences corresponding to an E2 protein(s) from an HPV, including especially, an E2 protein from a high risk HPV. The DNA binding domain and transcriptional activation domain of the E2 polypeptide can be one contiguous polypeptide chain, or in those embodiments where the E2 protein is directly formulated into the therapeutic composition, the DNA binding and transcriptional activation domain portions of the therapeutic E2 polypeptide can be provided as two separate peptide chains which have been chemically cross-linked, e.g., other than by a amide bond. The E2ad/db polypeptide can be a full length E2 protein, e.g., also including a hinge region sequence or the like, or can lack other E2 peptide sequences except for the DNA binding and transcriptional activation domains. The E2ad/db polypeptide may be derived from any species, e.g., human, bovine, rabbit, and from any papillomavirus subtype. In a preferred embodiment the E2ad/db has an alteration, for example, a E39A substitution or an altered hinge region, e.g., a deletion of residues corresponding to BPV E2xcex94220-309.
The subject method can be used to inhibit pathological progression of papillomavirus infection, such as preventing or reversing the formation of warts, e.g Plantar warts (verruca plantaris), common warts (verruca plana), Butcher""s common warts, flat warts, genital warts (condyloma acuminatum), or epidermodysplasia verruciformis; as well as treating papillomavirus-infected cells which have become, or are at risk of becoming, transformed and/or immortalized, e.g. cancerous, e.g. a laryngeal papilloma, a focal epithelial, a cervical carcinoma.
Another aspect of the present invention relates to a pharmaceutical preparation comprising a therapeutically effective amount of a recombinant transfection system for ameliorating a papillomavirus-induced condition in a subject. For instance, the transfection system, which is for gene therapy, includes a gene construct having a nucleic acid encoding an E2ad/db polypeptide and operably linked to a transcriptional regulatory sequence for causing expression of the E2 polypeptide in eukaryotic cells. The gene construct is provided in a gene delivery composition for delivering the gene construct to a papillomavirus infected cells and causing the cell to be transfected with the gene construct. For example, the gene delivery composition can be, e.g., a recombinant viral particle, a liposome, a poly-cationic nucleic acid binding agent, or a gene therapy vector derived from, e.g., a retrovirus, adeno-associated virus, or adenovirus.
Yet another aspect of the invention relates to a pharmaceutical preparation comprising a therapeutically effective amount of an E2ad/db polypeptide, formulated in the pharmaceutical preparation for delivery into PV-infected cells of an animal. In preferred embodiments, the polypeptide is formulated as a liposome.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).